HHV-8: A new herpesvirus associated with Kaposi's sarcoma

R E V I E W S

HHV-8: a new herpesv rus assocmted w th Kapos 's sarcoma Margaret K. Offermann Kaposi's sarcoma (KS) is a muhifocal aposi's sarcoma (KS) is Presence of HHV-8 in diverse neoplasm that develops in a high a mesenchymal tumor risk groups for KS proportion of HIV-infected and is the most frequent Recent studies with KS biophomosexual men. KS lesions contain neoplasm seen in individuals sies have identified novel DNA novel herpes-like DNA sequences with AIDS 1. Populations at risk sequences homologous to genes for KS include HIV-infected (HHV-8), and recent studies characterizing of the Gammaherpesvirinae, HHV-8 and its association with KS, homosexuals 2, patients who Herpesvirus saimiri and Epsteinand with other diseases, suggest that are pharmacologically immunoBarr virus (EBV) m. These DNA HHV-8 may represent the etiological suppressed to prevent transsequences have been found in viral pathogen. plant rejection 3, non-HIVdiverse types of KS, including HIV-associated 1°, classic11,12, infected individuals in areas of M.K. Offermann is in the Divn of Africa where HIV is endemic 4 African endemic ~3,~4,transplantHematology/Oncology, Dept of Medicine and and elderly men of Jewish or associated ~s and HIV-negative Winship Cancer Center, Emory Universi~,, Atlanta, Mediterranean ancestry s. Epihomosexual men '2. Originally GA 30322, USA. tel: +1 404 778 5808, demic or AIDS-associated KS called KS-associated herpesvirus fax: +1 404 778 5016, e-mail: [email protected] is aggressive with widespread (KSHV), the virus is now redissemination and frequent inferred to as human herpesvirus volvement of lymph nodes and viscera 6. Although 8, or HHV-8. The presence of HHV-8 in KS lesions from diverse risk groups from Europe ~,ls, Asia 16, HIV is thought to be an important cofactor for the Africa 13,~4and America 1°,12,17supports the hypothesis development of KS, the epidemiology suggests that another sexually transmitted agent is also involved. that HHV-8 is involved in the pathogenesis of all forms HIV-infected homosexuals and bisexuals have a of KS. This association is distinct from the episodic much higher incidence of KS (>20%) compared with identification of other viruses, such as cytomegaloHIV-infected hemophiliacs (1%) and other sub- virus is or human papilloma virus 19, that have previgroups of HIV-infected patients 2. KS is rarely seen ously been hypothesized to be responsible for KS. in HIV-infected children 7, and the occurrence of KS in women is associated with acquisition of HIV from Molecular characterization of HHV-8 bisexual men 2. Although the proportion of men Although the genome of HHV-8 was originally estiwith KS as their AIDS-defining illness has declined 2, mated, by contour-clamped homogeneous electric-field the lifetime occurrence of this tumor in homosexual electrophoresis, to be 270 kb (Ref. 20), Patrick Moore men diagnosed with AIDS is approximately 50% recently reported [{1996) 1st international Kaposi's (Ref. 8). Sarcoma Meeting, Los Angeles, CA, USA] that the long Immunosuppressive therapy to prevent transplanted unique coding region of HHV-8 is -139 kb. HHV-8 organ rejection predisposes to Kaposi's sarcoma. A has extensive co-linearity with Herpesvirus saimiri z°, recent retrospective study of kidney-transplant recipia T-lymphotropic virus that causes fatal lymphoproents reports that KS occurred in 1.6% of recipients, liferative disease in several non-human primates z~. with onset 6-124 months after transplantation and with HHV-8 and Herpesvirus saimiri contain genes ena male/female ratio of 2.25:1. Complete subsidence of coding proteins that have significant homology with the lesions occurred in many patients after reduction of cellular proteins, including cyclins, bcl-2 and G-proteinimmunosuppressive therapy 3. There is also an endemic coupled receptors2Z,2-L These viral genes could interform of KS in Africa, sometimes occurring in the abfere with normal cell signaling and contribute to the sence ofHIV (Ref. 4). This form affects women, as well transformation process. as men, but is not as aggressive as when accompanied by HIV infection. Although KS is rare in HIV-infected Association of HHV-8 with other diseases children, it represents 2-10% of childhood cancers In addition to KS lesions, HHV-8 has been detected in African children 9. The classic form of KS occurs in in clinically uninvolved skin from some KS patients, elderly men of Mediterranean and Jewish descent. Al- although at much lower levels than in the lesions themthough it is not associated with imnmnosuppression, selves 11. HHV-8 sequences were also detected in pathothere are subtle changes in immunological function s. logical skin lesions, such as squamous cell carcinoma In general, KS lesions, associated with the classic form, and actinic keratoses, in transplant patients who were occur on the legs or lower arms and rarely involve the immunosuppressed and did not display clinical evidence viscera. o f K S 24. However, the sample size of this study was

K

Copyright © 1996 Elsevier Science Ltd. All rights rcscrvcd. 0966 842X/96/$15.00 T R E N I ) S IN M I ( : t < O B I O L O < ; Y

383

V()I..

4

NO.

10

PII; S0966-842X(96)10060 3 ()(TTOBER 1996

REVIEWS

only small and other investigators have not detected HHV-8 in skin tumors of transplant patients 2s. HHV-8 is present at high copy number in most, if not all, primary effusion lymphomas (previously referred to as body-cavity-based lymphomas)2L This is a rare form of HIV-associated B-cell lymphoma that manifests itself as lymphomatous effusions, usually in the absence of any identifiable tumor mass. Unlike KS, the primary effusion lymphomas often, but not always, contain EBV in addition to HHV-8 (Refs 27,28). Multicentric Castleman's disease is a polyclonal lymphoid proliferation with vascular hyperplasia often accompanied by severe systemic signs. HHV-8 was detected in the lymph nodes of all HIV-infected and 7/17 (41%) of non-HIV infected patients with multicentric Castleman's disease 29. Approximately 75% of the patients with both multicentric Castleman's disease and HIV developed KS, by comparison with only 13% of non-HIV-associated multicentric Castleman's disease patients. This suggests that HHV-8 may be responsible for the high incidence of KS associated with this condition, especially in HIV-infected individuals. Cellular location of HHV-8 expression in cutaneous KS KS lesions contain complex mixtures of cell types: the spindle cell represents the predominant and characteristic cell type, but fibroblasts, microvascular endothelial cells, dendritic cells, extravasated erythrocytes and a variety of leukocytes are also typically present 3°. Examination of KS biopsies by polymerase chain reaction (PCR)-in situ hybridization revealed that both spindle cells and endothelial cells of nodular cutaneous KS lesions contain HHV-8 (Ref. 31), which supports the hypothesis that KS cells are of endothelial origin 32and that infection with HHV-8 is important in the pathogenesis of the disease. Standard in situ hybridization did not reveal detectable HHV-8, indicating that the virus load is probably very low. Examination of control tissues, including normal skin, angiosarcoma and skin nevus, showed no evidence of HHV-8 (Ref. 31). HHV-8 expression in body fluids HHV-8 can be detected in peripheral blood leukocytes of some, but not all, KS patients~L In addition, some patients with HIV who do not have clinical KS have detectable HHV-8 in their peripheral blood leukocytes, and its presence is associated with a higher rate of development of KS. Despite the presence of HHV-8 in blood, KS rarely occurs in patients who develop HIV as a consequence of blood transfusion z. Although one study failed to find HHV-8 in blood from normal individuals ~3, in another study HHV-8 was shown to be present in the peripheral blood leukocytes from about 10 % of normal non-HIV infected individuals, and a 101000-fold increase in HHV-8 load in HW-seropositive patients, compared with HIV-seronegative individuals, was reported 34. The disproportionate number of homosexual and bisexual men with KS has led to the hypothesis that it is a sexually transmitted disease a. In a retrospective, blind evaluation of semen collected from HIV-infected men in 1989 and 1990, HHV-8 sequences were found

"~'RILNI)S IN

MIC?I<()BI()L()(IY

384

in 21/33 (64%) of HIV-positive homosexual men and 0/30 healthy donors, using unnested PCR 3s. Using nested PCR to enhance sensitivity, 30•33 (91%) specimens from HIV-positive homosexual men and 7/30 (23 %) specimens from healthy donors were positive. In a five-year follow-up, 13 of these HHV-8-positive individuals developed KS. These data suggest a potential sexual route of transmission of HHV-8. Although several other groups have not detected HHV-8 in specimens from healthy semen-donors 17,2s, a recent study of healthy Italian men, without HIV or KS, supports a sexual mode of transmission of HHV-8. The ejaculates of 30/33 healthy men (91%) were found, by PCR, to contain HHV-8 (Ref. 36), whereas sequences were detectable in only 1/18 samples (5.5 %) of normal skin. These results were confirmed in a blind study. Although the frequency of positive samples was high in sperm, the amount of HHV-8 detected was low. Paolo Monini and colleagues used 3 5 - 4 5 cycles of amplification followed by an additional 30 cycles with nested primers, and the sperm sample signals required Southern hybridization with an oligonucleotide probe to detect the HHV-8 sequence. By contrast, HHV-8 could be detected by the use of ethidium bromide in skin samples from KS lesions that underwent the same number of rounds of amplification. Although the sensitivity of PCR predisposes to artifacts, these blind studies, employing semi-quantitative PCR, used m o r e DNA 34'36, more rounds of amplification 34--76or different primers for PCR 35than the studies that failed to detect HHV-8 in normal individuals. The discrepancies in virus prevalence may be a consequence of these technical differences or of geographic differences in the selection of normal patients. HHV-8 might be a prevalent virus found at very low copy number in blood and semen of normal individuals. The data suggest that the viral load increases in HIV-infected individuals and that this higher load is associated with a higher rate of clinical disease. Comparison of skin samples of normal and KS patients also suggests a change in tissue distribution because normal patients generally do not have detectable HHV-8 in skin, even when HHV-8 DNA can be detected in other sites 36. Cultured cells

Cells have been cultured from a variety of KS sites, including skin lesions 32,37,lung nodules 3s, and pleural and peritoneal fluid 39. Most of these cells express multiple endothelial markers 32,38,39. Nonetheless, they possess features that are distinct from other cell types found in the skin 3z. Some KS-derived spindle-cell lines retain HHV-8 expression through at least the eighth passage4°, but most cultured cells from KS lesions do not contain detectable HHV-8 (Refs 17,37). When cultures were derived from KS skin lesions that were documented to contain HHV-8, although viral sequence could be detected in some cultures at the second passage, no HHV8 sequence was detectable by the third passage 37. As a low-copy-number episomal virus, perhaps HHV-8 offers no growth advantage in culture and, therefore, is not maintained. Alternatively, the virus may be cytopathic to the cultured cells. Despite the lack of HHV-8, KS-

v o ,

4

No.

10

()(2T()BER

1996

REVIEWS

derived cells maintain many KS-like features and express and respond to a number of cytokines that are abnormally elevated in KS lesions, including oncostatin M (Ref. 41), interleukin-6 (Refs 42,43), basic fibroblast growth factor (bFGF) 44,45and TNFct (Refs 32,42,43). Studies with cultured cells suggest that the Tat protein of HIV may contribute to the increased risk of KS in H1Vinfected patients. Although not required for the development of KS, infection with the HIV virus increases the risk of KS at least 20 000-fold relative to the general population and 300-fold relative to patients who are immunosuppressed without HIV 2. The association with HIV is unlikely to result from direct infection of the KS cells with HIV 3s. Normal endothelial cells do not respond to Tat by proliferating; however, conditioned media from activated T-lymphocytes induce normal vascular cells to proliferate and develop spindle features, resembling KS cells, in response to Tat (Ref. 46). This suggests that leukocyte-produced cytokines may cooperate with HIV infection in the pathogenesis of KS. Tat amplifies the ability of bFGF to induce macroscopic KS lesions in nude mice, and increases the intensity of macroscopic angiogenesis, spindle cell growth and edema in-the lesions47. It remains to be determined whether there is any direct interaction between HIV and HHV-8. HHV-8 can be maintained in several primaryeffusion-lymphoma cell lines, including BC-1, BC-2 (Refs 22,26), and BCBL-1 (Ref. 28). These cells have high copy numbers of HHV-8. By contrast with BCBL-1 cells2s, BC-1 and BC-2 also contain EBV (Refs 22,26,48). Lytic growth of HHV-8 can be induced in BCBL-1 cells 28. Incubation of these cells with phorbol ester induced a 15-fold increase in HHV-8 viral DNA and a 50-70-fold induction of transcription. Electron microscopy revealed abundant 100-nm particles in - 5 - 1 0 % of the cells, and nuclease-resistant particles were released by lysis of the cells. Serological studies Serological tests for HHV-8 are being developed to assist in determining the prevalence and/or clinical status of HHV-8 infection. Serum from 32/48 (67%) of HIV-infected patients with KS possessed antibodies to a protein (p40) present in butyrate-induced BC-1 cells, by contrast with 7 / 5 4 (13%) of HIV-infected patients without KS48. Many of the KS patients who did not have detectable antibodies to p40 were, nonetheless, infected with HHV-8. BC-1 cells that were infected with HHV-8 did not express p40 protein unless they were pre-treated with butyrate, suggesting that p40 is an HHV-8-encoded protein that is expressed when cells are induced from the latent state to enter the lyric cycle. As most patients with KS had serum antibodies to this protein, this might reflect a higher level of viral activation in these individuals. Although seroreactivity to p40 cannot be used to extrapolate prevalence of the virus, efforts are under way to develop serological tests that will reflect all stages of infection. Conclusions A number of features make HHV-8 an attractive candidate for the etiological infectious agent in Kaposi's

TRENI,

,N

385

sarcoma. It has been detected in KS lesions from all risk groups around the world and has also been detected in the characteristic spindle cells and microvascular endothelial cells within KS lesions, but not within adjacent normal skin. It is detectable in a high percentage of semen and blood from patients at risk for KS, and patients with higher levels of HHV-8 in these body fluids develop KS at a significantly higher rate than patients at risk who do not have detectable HHV-8. The presence of antibodies to an HHV-8 antigenic peptide correlates with the presence of KS. However, despite the strong association of HHV-8 with KS, it has not been proven conclusively to be the etiological agent. As HHV-8 can be detected in the blood and semen of a number of individuals who do not have clinical KS, infection with the virus appears to be insufficient to cause disease. The virus is also associated with other diseases, most notably primary effusion lymphomas. Host factors of a pro-inflammatory nature appear to contribute to the pathogenesis of KS. It is hoped that the cloning of HHV-8 and the ability to grow HHV-8 in culture will allow studies to address the biological consequence of HHV-8 gene products and their interaction with other host and infectious factors. Acknowledgements I thank Dr Robin A. Weissfor criticalreviewof this manuscript.The author is supported,in part, by grants RO1CA60345, RO1CA67382 and P30AR42687 fromthe NIH. References 1 Kaplan,M.H. etal. (1987) Am. J. Med. 82, 389-396 2 Beral,V. et al. (1990) Lancet 335, 123-128 3 Montagnino,G. et aI. (1994)Am.J. Nepbrol. 14, 121-126 4 Bayley,A., Downing,R. and Cheingsong-Popov,R. (1985) Lancet 1,359-361 5 Friedman-Birnbaum,R. et al. (1993)Am.J. Dermatopathol. 15, 523-527 6 Gottlieb,G.J. and Ackerman,A.B. (1982) Hum. Pathol. 13, 882-892 7 Steeper,T.A. et al. (1992) Curt. Probl. Diagn. Radiol. 21, 79-109 8 Katz,M.H. et aI. (1994)J. Infect. Dis. 170, 198-202 9 Stiller,C.A. and Parkin, D.M. (1994) Paediatr. Perinat. Epidemiol. 8, 107-119 10 Chang,Y. et al. (1994) Science 266, 1865-1869 11 Dupin,N. et al. (1995) Lancet 345, 761-762 12 Moore,P.S. and Chang,Y. (t995) New Engl. J. Med. 332, 1181-1185 13 Chang,Y. etal. (1996) Arch. Intern. Med. 156,202-204 14 Schalling,M. et al. (1995) Nat. Med. 1,707-708 15 Boshoff,C. et al. (1995) Lancet 345, 1043-1044 16 8u, I.J. et al. (1995) Lancet 345, 722-723 17 Ambroziak,J.A. et al. (1995) Science 268,582-583 18 Ioachim,H.L. et al. (1992)Mod. Pathol. 5, 169-178 19 Huang,Y.Q. et al. (1992) Lancet 339, 515-518 20 Moore,P. et al. (1996)J. Virol. 70, 549-558 21 Ahuja,S.K.and Murphy,P.M. (1993)J. Biol. Chem. 268, 20691-20694 22 Cesarman,E. et al. (1995) Blood 86, 329A 23 Albrecht,J.C. et al. (1992)J. Virol. 66, 5047-5058 24 Rady,P. et al. (1995)Lancet345, 1339-1340 25 Weiss,R. (1996) Nat. Med. 2, 277-278 26 Cesarman,E. et al. (1995) New Engl. J. Med. 332, 1186-1191 27 Cesarman,E. et al. (1995) Blood 86, 2708-2714 28 Renne,R, et al. (1996) Nat. Med. 2, 342-346 29 Soulier,J. et al. (1995) Blood 86, 1276-1280

VOL

4

No.

10

OCTOBI%I

1996

R E V I E W S

30 Jones,R.R. (1986)Am.J. Dermatopatbol. 8, 369-370 31 Bosboff,C. et aI. (1995) Nat. Med. 1, 1274-1278 32 Yang,J. et al. (1994),1. Immunol. 152, 361-373 33 Whitby,D. etal. (1995) Lancet 346, 799-802 34 Bigoni,B. et al. (1996)J. Infect. Dis. 173,542-549 35 Lin,J-C. et al. (1995) Lancet 346, 1601-1602 36 Monini,P. et al. (1996) New Engl. J. Med. 334, 1168-1172 37 Lebbe,C. et al. (1995) Lancet 345, 1180 38 Salabuddin,S.Z. et al. (1988) Science 242, 430-433 39 Corbeil,J. et al. (1991)J. Immunol. 146, 2972-2976 40 Offermann,M. et al. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. (in press) 41 Miles,S.A.et al. (1992) Science 255, 1432-1434 42 Oxholm,A. et al. (1989) Acta Patho/. Microbiol. Scand. 97, 533-538

43 Yang,J., Hagan, M. and Offermann,M. (1994)J. Immunol. 152, 943-955 44 Scbulze-Osthoff,K., Goerdt,S. and Sorg,C. (1990)J. Invest. Dermatol. 95,238-240 45 Ensoli,B. et al. (1989) Science 243,223-226 46 Albini,A. et al. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 4838-4842 47 Ensoli,B. (1994) Nature 371,674-680 48 Miller, G. et al. (1996) New Engl. J. Med. 334, 1292-1297 Note added in proof There have been three new studiesthat strengthenthe evidencefor a link betweenKSand HHV-849-51. 49 Gao, S-J. etal. (1996) Nat. Med. 2, 925-928 50 Kedes,D. etal. (1996) Nat. Med. 2, 918-924 51 Chang,Y. et al. (1996) Nature 382, 410

Genetic engineering of animal RNA viruses KarI-Klaus Conzelmann and Gregor Meyers ecombinant DNA techThe ability to genetically manipulate recombinant RNA has reprenology makes it possviruses has led to extraordinary advances sented a major technical probible to specifically in understanding virus biology and to the lem and was achieved only two modify DNA genomes. Because establishment of useful vector systems. years ago. In this review, basic of the small size of their genInitially confined to DNA viruses and features of RNA virus gene exomes, viruses are particularly retroviruses, RNA viruses have more pression, methods for genetic amenable to such manipularecently become attractive candidates manipulation of RNA viruses, tions. Since the recovery of the for expression of heterologous genes and and perspectives of RNA virusfirst recombinant virus, simian offer promising perspectives for based expression systems are virus 40 (SV40), 20 years ago I, biomedical applications. described. a variety of DNA viruses and K-K. Conzelmann* and G. Meyers are in the retroviruses has been genetiGene expression of positive Dept of Clinical Virology, Federal Research Centre cally manipulated to exp~loit strand RNA viruses for Virus Diseases of Animals, Paul-Ehrlich-Str. 28, their replication machinery' for The plus-stranded RNA viruses D-72076 Tiibingen, Germany. expression of heterologous were the first that were open to *tel: +49 7071 967 205, fax: + 4 9 7071 9 6 7 303, e-mail: [email protected] genes. When transfected into direct genetic manipulation s-v a cell, the purified DNA of because their genomic RNA many of these viruses is infectious and gives rise to (vRNA) is able to function as an mRNA, directing the new viral particles. Alternatively, recombinant DNA production of all viral proteins necessary for the initican be introduced into virus DNA by homologous ation of virus propagation. To provide a template for recombination with the genome of a helper virus, the synthesis of additional mRNA molecules, replication allowing the manipulation of large DNA viruses, starts with the polymerization of a minus strand such as herpes 2 or pox viruses -~,4.In recent years, RNA complementary to the genome (cRNA). Thus, for all viruses lacking a DNA phase in their replication cycle positive-strand RNA viruses the components of the have also been manipulated. Since recombinant RNA replicase complex have to be translated directly from techniques remain cumbersome, almost all studies the genomic RNA. For the other viral polypeptides, with RNA viruses have relied upon cDNA intermediwhich mainly constitute structural proteins, two ates to produce biologically active RNA molecules. principle expression strategies exist, by which positiveTwo major RNA virus groups, the positive strand and strand RNA viruses can be classified into two groups the negative strand RNA viruses, are distinguished (Fig. 1). based on whether their purified RNAs are able to inViruses in the first group generate only one kind of itiate an infectious cycle or not. For the latter group, mRNA, which is of genome length and contains one the minimal infectious unit is not an RNA molecule long open reading frame (ORF). Expression of all viral but a ribonucleoprotein qomplex (RNP). The reproteins is achieved by translation of this RNA into constitution of functional RNPs from proteins and a polyprotein that is co- and post-translationally

R

Copyright © 1996 Elsevier Science Ltd. All rights reserved. 0966 842X/961515.00

TRFNI'~S

IN MI(:I'-.OI~I<)LO(;Y

386

V()L.

4

No.

IO

P[I: SO966-842X(96)10062-7

(~)(;'IOBt~t~.

1996